The present invention relates to antagonistic prokaryotic/eukaryotic associations, and particularly to the formation of associations of eukaryotic organisms with L-form bacteria antagonistic to a secondary organism with the effect of conferring the antagonism on the eukaryote.
L-form bacteria are bacteria without cell walls which can be induced by suppressing cell wall synthesis with agents such as penicillin (Madoff, 1986, Introduction to the Bacterial L-forms in xe2x80x9cThe Bacterial L-formsxe2x80x9d. Marcel Decker, New York, pp 1-20). The artificial production of intracellular associations of L-forms with plants was first described by Aloysius and Paton (1984); Artificially Induced Symbiotic Associations of L-form Bacteria and Plants. Journal of Applied Bacteriology 56, 465-477). A variety of procedures are now available to achieve such associations (Paton and Innes, 1991. Methods for the establishment of Intra-cellular Associations of L-form bacteria and higher plants. Journal of Applied Bacteriology 71, 59-64).
In our EP-0136035 there are described associations of prokaryotic organisms and eukaryotic plant cells produced by bringing their L-form into contact with the plant cell. The scientific principles underlying the practical applications of the present invention have as their starting point the said European Patent disclosure which reveals that L-form prokaryotes, produced from bacteria with entire cell walls, can be induced to enter and become viably established in eukaryotic organisms such as angiosperms.
Various methods for the preparation of L-form inocula for this purpose are available e.g. by the isolation and culture of pure L-forms (stable or unstable) or by using a heterogeneous mixture of L-forms and cell-walled bacteria formed by the introduction of an inducing agent into a fresh suspension of bacteria in a protective osmoticum. It has also been revealed that plant associations with L-form bacteria can be created using such inocula by a variety of procedures, for example, by contacting germinating seed directly with L-form containing inocula whereby the root system is infected leading to further passage of the organism within the plant or by direct invasion of the plant tissue using, for example, hypodermic inoculation techniques (Paton, (1987) Journal of Applied Bacteriology 63, 365-371).
We have now found that if the eukaryotic host and the prokaryotic bacteria are correctly chosen, it is possible to achieve not just protection of the host from subsequent attack by the walled form of the L-form bacteria, although this may occur, but also protection of the host against quite different invasive organisms to which the bacteria is antagonistic. It is thus possible in the first aspect of the invention to provide means for protecting a host plant from infection by an invasive organism unrelated to the associated prokaryote, by selecting a prokaryote which is both capable of forming an L-form association with the host eukaryote, and which is antagonistic to a selected invasive organism.
It is also possible to select an association of L-form prokaryote and eukaryotic host in which the L-form prokaryote has the effect of protecting the host plant against the entire forms of the selected L-form prokaryote, and also protecting the plant and/or fruits thereof or other commercially valuable parts of the plant against invasive agents to which the L-form prokaryote selected is antagonistic.
There is no prior art of which we are aware that reveals that plant disease can be controlled by utilising associations of L-form bacteria antagonistic to selected plant pathogens since previous disclosures indicate that the protection can be produced against walled form of a bacterial pathogen due to a plant reaction to the presence of the L-form of that pathogen. (Amijee et al. (1992); Biocontrol Science and Technology 2 203-214.
As has already been established in our European Patent a wide variety of associations have been achieved and we have found as yet, no restriction to this. Innumerable bacteria antagonistic to either or both bacterial and fungal plant pathogens are known to exist in nature and are important factors in the preservation of the ecological equilibria. Any of these can be selected for a desired antagonistic activity and thereafter its effect on the resistance of the plant may be determined. Our procedure offers therefore a wide range of possible options along with selection on the basis of desired activity, safety and ethical considerations.
According therefore to a first feature of the invention there is provided a process for protecting a host eukaryote or a part thereof, from attack by an invasive agent, which comprises; selecting a prokaryotic bacteria which is capable of forming an L-form association with said host, and introducing said bacterium into the said host; the invention being characterised in that said bacterium is also selected to be antagonistic to the invasive agent.
In a preferred embodiment of the invention the selected L-form bacterium is also selected to give resistance to the host against the entire forms of said bacterium. The introduction of the L-forms into the host eukaryote may be effected by:
1. injection of the host with a culture of an entire bacterium along with an effective amount of an L-form inducer and an osmoticum, or
2. by macerating eukaryotic host tissue which is infected with the L-form bacteria, and bringing the macerate or a filtrate thereof into contact with susceptible cells of the eukaryotic host;. or
3. by forming an osmotically compatible aqueous suspension of the L-form bacteria and bring the said suspension, or a filtrate thereof, into contact with susceptible cells of the eukaryotic host.
The host may be an angiosperm, a monocotyledonous or dicotyledonous plant. The invasive organisms may be prokaryotes or eukaryotes, for example, bacteria or fungi and may be pathogens or saprophytes.
This invention is useful for the protection of plants and their constituent parts including vegetative extensions and fruits, and thus provides, for example, an ecologically and ethically acceptable method of prolonging the shelf life of a soft fruit such as the strawberry or raspberry and vegetables such as Chinese cabbage and tomatoes. Further, some antagonistic bacteria such as, for example. Pseudomonas antimicrobica have a broad spectrum activity against both fungi and bacteria and hence protect host eukaryotes against undesired organisms. The invention thus seeks to provide a means of controlling disease both pre- and post-harvest so that the interest of the grower and crop utiliser can be achieved by the same protective treatment.
According therefore to the second aspect of the invention there is provided a eukaryotic plant or part thereof comprising L-form prokaryotic bacteria which bacteria are antagonistic to a selected invasive agent. The L-form prokaryotic bacteria may also protect the plant against entire forms of the bacteria selected. The plant may be a soft fruit such as the strawberry or raspberry, black current, red current or a vegetable such as Chinese cabbage, tomato, courgette, bean or potato. In one preferred opinion, when the soft fruit is a strawberry, the L-form bacteria may be Pseudomonas antimicrobica. Similarly Pseudomonas antimicrobica and Erwinia herbicola will protect the Chinese cabbage both during growth and against deterioration after harvest.